The proposed work will examine the cellular basis for regulation of contractile force in heart muscle. This study will examine how specific fundamental cellular mechanisms vary during the cardiac cycle and influence force production. Quantitative examination of the following cellular features will be carried out and their influence on the generation of force assessed: (1) The intracellular activity of three important cations - sodium (aiNa), calcium (aiCa) and hydrogen (pHi); (2) The trans-sarcolemmal movement of calcium and sodium (via the calcium current, the sodium current, the Na/Ca exchanger and the Ca-activated non-selective cation current); (3) The internal stores of calcium (the sarcoplasmic reticulum). This work will be carried out in ventricular muscle and in cardiac Purkinje fibers over five years. Single ventricular muscle cells from rat and guinea pig (enzymatically dissociated) will be voltage-clamped using a single-microelectrode method. A fluorescent indicator will be used to measure aiCa (fura-2), aiNa will be measured using an ion-selective microelectrode and pHi will be measured using both techniques (with BCECF as the optical indicator). Video imaging techniques will be used to measure sarcomere length, cell length and the spatial distribution of intracellular calcium. Sheep Purkinje fibers will be examined using a two-microelectrode voltage-clamp method while measuring tension, aiCa (using the calcium-activated photoprotein aequorin), and aiNa and pHi (using ion-selective microelectrodes). This combination of new techniques and established methods will permit us, for the first time, to examine quantitatively the ionic control of tension in myocardium and in Purkinje fibers under normal conditions and when resting aiCa is elevated ("calcium overload"). The planned experiments will address the following questions: (1) What mechanisms control resting aiCa? How important is the mean level of aiNa in controlling aiCa? (2) How is the peak level of aiCa (during the twitch) regulated and how is it modulated by aiNa? (3) How is peak aiCa related to the resting aiCa? How is this relationship altered during "calcium overload"? (4) How do changes of heart rate and of action potential duration modify the answers to these questions? (5) How do therapeutic agents (e.g. cardiotonic steroids, blockers or Na+ and Ca++ channels) modify the above relationships? The proposed research should broaden our understanding of the cellular mechanisms that govern the development of tension in heart. By revealing the physiological control of contraction in heart and its modification by therapeutic agents, this work should lay the foundation for improved treatment of diverse cardiac disease including heart failure.